Project Summary/Abstract: Acute and chronic pain is associated with disturbed sleep that leads to enhanced pain sensitivity. However, less is known of the specific neural pathways that cause arousal in response to painful stimuli. Anatomical studies have shown that the spino-parabrachial circuit relays nociceptive signals from the spinal cord. The lateral PB, and especially the external lateral part of the PB (PBel), receives nociceptive spinal afferents from numerous spinal neurons and then relays to forebrain and brainstem sites. Most of the PBel neurons express the calcitonin gene-related peptide (CGRP), and we have recently shown that these neurons in the PBel regulate waking-up response to hypercapnia by projecting to forebrain arousal areas. Based on our recent study and anatomy known so far, we hypothesize that PBelCGRP neurons is also the critical relay node that transmits pain to induce cortical arousals from sleep. We will test this hypothesis in Aim1, by inhibiting PBelCGRP neurons using optogenetic and chemogenetic tools and investigate if PBelCGRP neurons are important to waking up to pain, in both opto-pain model (activating CGRP nociceptors in foot) and in the conventional inflammatory-pain model. Our preliminary data suggests that blocking PBelCGRP neurons prevents waking up to CGRP nociceptor stimulation. In Aim2, we will selectively block each of the terminal fields of PBelCGRP neurons while activating the CGRP nociceptors to induce acute pain stimuli. We have used this strategy recently to unravel the pathways regulating waking up to CO2. In Aim2, we will investigate which one of its projection targets, such as the substantia innominata (SI), the lateral hypothalamus (LH), the central nucleus of the amygdala (CeA), and the bed nucleus of the stria terminalis (BST) are critical and most effective in blocking pain induced wakefulness. Finally, in Aim3, using Channel Rhodopsin Assisted Circuit Mapping experimental approach, we will first establish, if PBelCGRP neurons directly excite BF, CeA, BNST and LH neurons in the brain slices, and next, we will test the functional synaptic connectivity between dorsal horn (DH) neurons in spinal cord and PBelCGRP neurons (DH ? PBelCGRP). Thus, modulating both peripheral nociceptors and central pathways in a same mouse will help us to selectively dissect the role of PBelCGRP neurons and their outputs pathways in regulating pain induced arousals. Investigating this converging neuro-circuitry for regulating pain and arousal will help us understand their interaction and pave way for designing novel therapeutic strategies targeted to the non-opioidergic pathways that can effectively block pain induced sleep disturbances, without limitation of developing tolerance and abuse potential.